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Characterization of a Novel Intracellular Endopeptidase of the /ß Hydrolase Family from Streptomyces coelicolor A3(2)

István Nagy,^1,2 Tisha Banerjee,¹ Tomohiro Tamura,^2, Geert Schoofs,¹ Ann Gils,³ Paul Proost,⁴ Noriko Tamura,^2, Wolfgang Baumeister,² and René De Mot^1*

Centre of Microbial and Plant Genetics,¹ Laboratory for Pharmaceutical Biology and Phytopharmacology,³ Laboratory of Molecular Immunology, Rega Institute for Medical Research, Catholic University of Leuven, Leuven, Belgium,⁴ Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany²

Received 3 June 2002/ Accepted 21 October 2002

In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsin-like activity, absent from the wild type, was detected. Complementation that restored proteasome function did not suppress expression of the endopeptidase. Since the enzyme was not found in two other S. coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant. Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S. coelicolor A3(2) genome with no known homologue. Based on the prediction of a Ser-Asp-His catalytic triad and an /ß hydrolase fold, SCO7095 was assigned to peptidase clan SC. N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization. Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected. On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.

Present address: National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan.

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